Psychopharmacologically active d-glu or d-his containing peptides

ABSTRACT

1. A PEPTIDE OF THE CORMULA:   A-GLU(Q)-HIS-X   IN WHICH ONE OF THE AMINO RADICALS GLU(Q) OR HIS IS PRESENT IN THE D-FORM; IN WHICH A IS SELECTED FROM THE GROUP CONSISTING OF H-L-MET, H-L-MET($O), H-L-MET($O2) DESAMINO-MEL, DESAMINO-MET($O), DESAMINO-MEL($O2), AND THE MOIETY; H2N-B-CO- IN WHICH B IS ALKYLENE HAVING 1-6 CARBON ATOMS; Q IS SELECTED FROM THE GROUP CONSISTING OF OH AND NH2; X IS SELECTED FROM THE GROUP CONSISTING OF HYDROXY; (N-PHENYLALKYL) AMINO OF THE FORMULA   -NH-ALK-PHENYLENE-R1   IN WHICH ALK IS ALKYLENE WITH 1-6 CARBON ATOMS, AND R1 IS SELECTED FROM THE GROUP CONSISTING OF HYDROGEN AND HYDROXY; AND -L-PHE-Y; Y IS SELECTED FROM THE GROUP CONSISTING OF HYDROXY, DESCARBOXYL-LYSYL, DESCARBOXY - ARGIINYL, -L-LYS-Z, -L-ARG-Z, -D-LYS-Z, AND -D-ARG-Z; Z IS SELECTED FROM THE GROUP CONSISTING OF HYDROXY, (N-B-INDOLYL-ETHYL) AMINO, -L-TRP-OH,   -L-TRP-GLY-OH,   -L-PHE-OH, -L-PHE-GLY-OH, AND (N-PHENYLALKYL) AMINO OF THE FORMULA   -NH-ALK-PHENYLENE-R1   IN WHICH ALK IS ALKYLENE WITH 1-6 CARBON ATOMS, R1 IS SELECTED FROM THE GROUP CONSISTING OF HYDROGEN AND HYDROXY; AND FUNCTIONAL DERIVATIVES OF SAID PEPTIDE SELECTED FROM THE GROUP CONSISTING OF PHARMACEUTICALLY ACCEPTABLE ACID ADDITION SALTS DERIVATIVES IN WHICH ONE OR MORE FREE AMINO GROUPS ARE SUBSTITUTED BY ACYL DERIVED FROM AN ALIPHATIC CARBOXYLIC ACID WITH 1-6 CARBON ATOMS, UNSUBSTITUTED AMIDES OR LOWER ALKYL (1-6C) SUBSTITUTED AMIDES OF THOSE PEPIDES HAVING A FREE CARBOXYL GROUPS, ESTERS DERIVED FROM ALIPHATIC OR PHENYLALIPHATIC ALCOHOLS WITH 1-18 CARBON ATOMS, AND METAL COMPLEXES THEREOF.

United States Patent 3,850,904 PSYCHOPHARMACOLOGICALLY ACTIVE D-GLU 0RD-HIS CONTAINING PEPTIDES Hendrick Marie Greven, Heesch, Netherlands,assignor to Akzona Incorporated, Asheville, N.C.

No Drawing. Filed May 14, 1973, Ser. No. 359,923 Claims priority,application Netherlands, May 24, 1972, 7206941 lint. Cl. A61k 17/06.27/00; C07c 103/52 US. Cl. 260-1125 Claims ABSTRACT OF THE DISCLOSUREThe invention relates to psychopharmacologically active peptides andpeptide derivatives of the general formula:

in which one of the amino acid radicals Glu(Q) or His is present in theD-form, and in which A represents: L-Met, L-Met(- O), L-Met(- Odesamino-Met, desamino-Met( 0), desamino-Met(- O or the moiety: HN-B-Co-, in which B is a branched or unbranched alkylene group with 1-6carbon atoms,

Q represents: the group OH or NH and X represents: a hydroxyl group, a(N-phenylalkyl)amino group, or the group L-Phe-Y, in which Y is ahydroxyl group, a (N-arninoalkyl)amino group, or the group L-Lys-Z,D-Lys-Z, L-Arg-Z, or D-Arg-Z, in which Z is a hydroxyl group, the group(N-fi-indolylalkyDamino, or the group L-Trp-OH, the group L-Phe-OH, a(N- phenylalkyl)amino group, in which the alkyl group contains 1-6carbon atoms, the group L-Phe-Gly-OH or the group -L-Trp-Gly-OH,

as well as the functional derivatives thereof.

The above peptides and peptide derivatives inhibit the extinction ofconditioned avoidance response, that means that they can be used for thetreatment of mental disorders, whereby a stimulation of the mentalperformance is desired.

The invention relates to peptides and peptide derivatives with highlyactive psychopharmacological properties.

From European Journal of Pharmacology 2, 14 (1967) certain peptidefragments of the natural adrenocorticotrophic hormones (ACTH) are knownto inhibit the extinction of the conditioned avoidance response.Especially the peptide having the amino acid sequence 1-10 of ACTHproved to be active in this respect. Moreover it was found that thefirst three amino acids (Ser-Tyr-Ser) could even entirely be omittedwithout much loss of activity. The article ends with the conclusion thatthe pertide with the amino acid sequence 4-10 of ACTH, vizH-Met-Glu-His-Phe-Arg-Trp-Gly-OI-I, is the shortest peptide, and perhapsthe key sequence for the said activity.

The peptide with the amino acid sequence 4-10 of ACTH does not onlyexert the psychopharmacological property mentioned above, but also aslight MSH activity, as usual in this type of ACTH fragments. Althoughthe eflect of a low dose administration of a MSH active peptide in menis still unknown, a search was done for peptides having at least thesame psychopharmacological activity but no or a reduced MSH-activity.

In my co-pending Netherlands patent application 7202278 andcorresponding U.S. application Ser. No. 331,945, filed Feb. 12, 1973, itis already disclosed that the N-terminal amino acid L-Met of theoriginal 4-10 ACTH peptide can be replaced without any loss of activity,i.e. by L-Met( 0), L-Met( O desamino- Met, desamino-Met( 0),desamino-Met( O or the 3,850,904 Patented Nov. 26, 1974 group: H N-B-CO-in which B represents: a branched or unbranched alkylene group with 16carbon atoms, such as glycyl, valyl, alanyl, fl-alanyl ora-methylalanyl.

Furthermore the co-pending Netherlands patent application 7203042 andcorresponding US. application Ser. No. 337,507, filed Mar. 2, 1973,describes that the replacement of the C-terminal peptide radical-L-Trp-Gly- OH of the original 4-10 ACTH peptide by a group selectedfrom L-Phe-OH, L-Phe-Gly-OH and the group (N-phenylalkylamino-moiety)causes a considerable increase of the phychopharmacological activity.

Moreover the co-pending Netherlands patent application 7204422 statesthat a considerable increase of the psychopharmacological activity isobtained by replacing the amino acid L-Arginine or L-Lysine of theoriginal 4-10 ACTH-peptide with the corresponding D-amino acid.

Surprisingly, it has now been found that an increase of thepsychopharmacological activity can also be effected by replacing theamino acid radical L-Glu(Q) or L-His of the original 4-10 ACTH-peptide,or of any of the modified peptides, described in the co-pending Dutchand U.S. patent applications mentioned above, with the correspondingD-arnino acid radical.

The present invention therefore comprises the manufacture and use ofpeptides and peptide derivatives of the general formula:

A-Glu (Q) -His-X I in which one of the amino acid radicals Glu(Q) or Hisis present in the D-form, and in which A represents: H-L-Met,H-L-Met(+0), HL-Met( O desamino'Met, desamino-Met( 0), desamino-Met- Oor the moiety: H N-B-CO, in which B stands for a branched or unbranchedalkylene group with 1-6 carbon atoms,

Q represents: a hydroXyl or an amino moiety, and

X represents: a hydroxyl group, a (N-phenylalkyl)amino group, in whichthe alkyl group possesses 16 carbon atoms, or the group L-Phe-Y, inwhich Y represents a hydroxyl group, a (N-aminoalkyl)-amino group, thealkyl group of which possesses- 2-6 carbon atoms, or the group L-Lys-Z,D-Lys-Z, L-Arg-Z or D-Arg-Z, in which Z is a hydroxyl group or a(N-fi-indolylalkyl) amino group, the alkyl group of which may contain1-6 carbon atoms, or the group L-Trp-OH, L-Phe-OH, a(N-phenylalkyl)amino group, the alkyl group of which contains 1-6 carbonatoms, the group L-Phe-Gly-OH or L-Trp-Gly-OH,

as Well as the functional derivative thereof.

The above-mentioned non-amino acid residues: (N- phenylalkyl)arnino,(N-aminoalkyDamino and (N-fldndolylalkyl)amino, are moieties that arechiefly distinguished from amino acid radicals by the absence of thecarboxyl group.

By a (N-phenyla1ky1)amino group is meant in this connection a moiety ofthe general formula:

in which Alk represents a branched or unbranched alkylene group with 1-6carbon atoms, and R stands for hydrogen, halogen, hydroxy or an alkyl oralkoxy group with 1-4 carbon atoms.

By a (N-aminoa1kyl)amino moiety is meant a group of the general formula:

-NA1k-N in which Alk represents a branched or unbranched alkylene groupwith 2-6 carbon atoms, R represents hydrogen or alkyl with 14 C-atoms,and R hydrogen, alkyl with 1-4 carbon atoms, or an amidine group.

A (N-B-indolylalkyDamino group is a moiety of the general formula:

-NAlk-- H L in which Alk represents a branched or unbranched alkylenegroup with 1-6 carbon atoms.

By replacing one of the amino acid radicals L-Glu or L-His of theoriginal 4-10 ACTH peptide with the correspnding D-Glu or D-His aminoacid radical the psychopharmacological activity observed generally risesby factor 3.

This modification introduced into the peptides described in theaforesaid Dutch co-pending patent applications causes a further increaseof activity.

Special preference is given to peptides according to the invention inwhich the amino acid histidine occurs in the D-form, whether or notincombination with other modifications known from my co-pending patentapplications.

Especially the following peptides were found to have potentpsychopharmacological activities:

desamino-Met-L-Glu-D-His-L-Phe-L-Arg( or L-Lys)-L- Phe-OH,

H-L-Met -L-Gln-D-His-L-Phe-L-Arg (or L-Lys) Tra,

H-L-Met-L-Glu-D-His-L-Phe-L-Arg(or L-Lys)-Tra,

H-L-Met O) -L-Glu-D-His-L-Phe-L-Arg (or L-Lys L-Phe-OH,

H-L-Met-D-Glu-L-His-L-Phe-L-Arg (or L-Lys -L-Phedesamino-Met-L-Glu-D-His-L-Phe-L-Arg(or L-Lys) -Tra and thecorresponding sulfoxide and sulfone, H-L-Met-L-Glu-D-His-L-Phe-D-Lys-L-Phe-OH and the corresponding sulfoxide andsulfone.

The peptides and peptide derivatives according to the invention areprepared by a process commonly used in peptide-chemistry. The processesthat are employed usually for the manufacture of the present compoundscan be summarized as follows:

(a) condensation of a compound (amino acid, peptide) having a freecarboxyl group and protected other reactive groups, with a compound(amino acid, peptide or amine) having a free amino group and protectedother reactive groups, in the presence of a condensation agent;

(b) condensation of a compound (amino acid, peptide) having an activatedcarboxyl group and optionally protected other reactive groups, with acompound (amino acid, peptide, amine) having a free NH group andoptionally protected other reactive groups;

(c) condensation of a compound (amino acid, peptide) having a freecarboxyl group and optionally protected other reactive groups, with acompound (amino acid, peptide, amine) having an activated amino groupand optionally rotected other eastive gr ups, A

after which the protecting groups are removed, if necessary.

Activation of the carboxyl group can be effected, for example, byconverting the carboxyl group into an acid halide, an azide, anhydride,imidazolide, or an activated ester such as the N-hydroxy-succinimidoester, or the pnitro-phenyl ester.

The amino group can be activated by converting it into a phosphite amideor by the phosphor-azo method.

Methods usually employed for the above condensation reactions are: thecarbodiimide method, the azide method, the mixed anhydride method andthe method of the activated esters as described in The Peptides, vol. I,1965 (Acad. Press), by E. Schroder and K. Liibke. Moreover Merrifieldsso-called Solid Phase Method, described in J. Am. Chem. Soc. 85, 2149(1963), can be applied for the manufacture of the present peptides andpeptide derivatives.

The reactive groups that are not allowed to participate in thecondensation reaction are protected effectively by the so-calledprotecting groups, which can be easily removed again, for example, byhydrolysis or reduction. Thus, for example, a carboxyl group can beprotected effectively by esterification with methanol, ethanol, tertiarybutanol, benzylalcohol or p-nitrobenzylalcohol, or by conversion into anamide. This latter protecting group is very hard to remove, however, sothat it is recommendable to use this group only to protect the carboxylgroup of the C-terminal amino acid in the ultimate peptide or the'y-carboxyl group of glutamic acid. In this case the peptide synthesisleads direct to the amide of a peptide according to formula I.

Groups that are capable of protecting an amino group effectively areusually acid groups, for example an acid group derived from analiphatic, aromatic, araliphatic or heterocyclic carboxylic acid such asacetic acid, benzoic acid, or pyridine-carboxylic acid, or an acid groupderived from carbonic acid such as the group ethoxy-carbonyl,benzyloxy-carbonyl, t-butyloxy-carbonyl orp-methyloxy-benzyloxy-carbonyl, or an acid group derived from a sulfonicacid, such as the group benzene-sulfonyl or ptoluene-sulfonyl, but alsoother groups can be employed, such as substituted or unsubstituted arylor aralkyl groups, for example benzyl and triphenylmethyl, or groupssuch as ortho-nitro-phenyl-sulfenyl and 2-benzoyl-1-methylvinyl.

It is mostly recommendable also to protect the guanidine group ofarginine, the e-amino group of lysine, and the imidazole group ofhistidine, but this protection is not absolutely necessary. Conventionalprotecting groups in this connection are a tertiary butyloxy-carbonyl,or a tosyl group for the e-amino group of lysine, a nitro group for theguanidine group of arginine, and a benzyl, dinitrophenyl or a tritylgroup for the imidazole group of histidine.

The protecting groups can be split off by various conventional methods,depending upon the nature of the group in question, for example withtrifluoro acetic acid, or by mild reduction, for example with hydrogenand a catalyst, such as palladium, or with HBr in glacial acetic acid.

Peptides according to the present invention having as the N-terminalmoiety a methionylsulfoxide or desaminomethionylsulfoxide group, may beprepared from the corresponding Metor Desamino-Met peptide by means of amild oxidation known per se, for example with dilute hydrogenperoxide ora peracid. This oxidation yields a mixture of the S- and R-sulfoxide(:dor l-sulfoxide), which mixture may be separated into the separatediastereomeric compounds in a conventional manner.

By coupling the S- or R-sulfoxide (=dor l-sulfoxide) of methionine ordesaminomethionine with the peptide H-Glu(Q)-HiS-X, in which eitherGlu(Q) or His occurs in the D-form and Q and X have the meanings 5indicated above, the separate enantiomers can also be obtained in adirect way.

The peptides according to the invention having as the N-terminal residuea methionylsulfone (Met or desaminomethionylsulfone (desamino Met- 0group may be prepared most conveniently by an oxidation known per se ofthe corresponding Metor Desamino-Met peptide, for example with H 0 or aperacid.

By functional derivatives of the peptides and peptidederivativesaccording to the invention are meant:

(I) The pharmaceutically acceptable acid addition salts of the peptidesand peptide derivatives,

(2) Peptides or peptide derivatives in which one or more free aminogroups have been substituted by an acyl group derived from an aliphaticcarboxylic acid with 1-6 carbon atoms, such as an acetyl group,

(3) Unsubstituted amides or lower alkyl (l6 C) substituted amides ofthose peptides and peptide derivatives according to the invention havinga free carboxyl group,

(4) Esters of the present peptides derived from aliphatic or araliphaticalcohols with 1-l8 carbon atoms; in particular, the lower aliphatic (l6C) alcohols, such as methanol, ethanol, butanol, pentanol orcyclohexanol, and the lower araliphatic (7-10 C) alcohols, such asbenzylalcohol, phenylethylalcohol, phenylpropylalcohol, 0rcinnamylalcohol,

Metal complexes formed by contacting the peptides or peptide derivativeswith a sparingly soluble salt, hydroxide or oxide of a metal, preferablyzinc, or preparations obtained by associating the present peptides withorganic, mostly polymeric, compounds, such as gelatine,polyphloretinphosphate or polyglutamic acid, to obtain a prolonged modeof action.

The acid addition salts are obtained by reacting the present compoundswith a pharmaceutically acceptable organic or inorganic acid, such asHCl, phosphoric acid, acetic acid, maleic acid, tartaric acid, or citricacid.

As already briefly said the present peptides and peptide derivatives aswell as their functional derivatives defined above have valuablepsychopharmacological activities. The present compounds inhibit theextinction of conditioned avoidance response, that means that they canbe used, in general, as antidepressant agents. More particularly theycan be used for the treatment of certain mental disorders whereby astimulation of the mental performance is desired, such as in certaintypes of neurosis and in old-age infirmities (senility).

The compounds according to the invention can be administeredparenterally, orally, sublingually, rectally or intranasally. Preferablythe peptides are placed in a form suitable for parenteraladministration, for which purpose they are dissolved, suspended oremulsified in a suitable liquid. However, mixed with suitableauxiliaries and/or fillers the peptides may also be placed in a formsuitable for oral, sublingual, rectal or intranasal administration.

The peptides or peptide derivatives according to the invention arepreferably administered parenterally in a 6 daily dosage of from 0.01 g.to g. per kg. body weight, dependent upon the peptides activity level.For oral, sublingual, rectal or intranasal administration the dailydosage may be considerably higher, preferably from 0.1 mg. to 100 mg.per kg. body weight.

Exceedingly valuable preparations are obtained if the present peptidesare placed in a form in which they have a prolonged activity, forexample, incorporated into gelatin, polyphloretinphospate orpolyglutamic acid, or preferably as metal complexes. These metalcomplexes can be obtained by contacting the peptides with sparinglysoluble metal salts, metal hydroxides or metal oxides. As sparinglysoluble metal salts the metal phosphates, metal pyrophosphates and metalpolyphosphates are commonly used.

Metals that can be used in this process are the metals belonging to theb-groups of the periodic system, for example cobalt, nickel, copper,iron, and preferably zinc, as well as the metals belonging to the maingroups of the periodic system and capable of forming complexes, such asmagnesium and aluminum. The preparation of the said metal complexestakes place in the conventional manner.

Thus, for example, a metal complex can be obtained by adding the peptideand a poorly soluble metal salt, metal hydroxide or metal oxide to anaqueous medium. The metal complex can also be obtained by adding analkaline medium to an aqueous solution of the peptide and an insolublemetal salt to form the insoluble peptide-metal hydroxide complex.

Moreover, the metal complex can be obtained by adding the peptide, asoluble metal salt and a soluble salt to an aqueous, preferably alkalinemedium to form an insoluble peptide/metal salt complex in situ.

The metal complexes can be employed at once as suspensions, or forexample be lyophilized and afterwards suspended again.

Biological activity.Extinction of the conditioned avoidance responseMale white rats weighing approximately grams were conditioned by meansof the so-called pole-jumping test. The conditioned stimulus was. alight presented over the cage for 5 seconds, whereupon the unconditionedstimulus of shock was delivered through the grid floor of the cage.

For 3 consecutive days 10 tests were run every day with an averageinterval of 60 seconds. The day after this acquisition period theextinction was studied in sessions of 10 trials. All animals that made 8or more positive responses in the first extinction session were treatedwith the substance to be tested or with a placebo. After that,extinction sessions of 10 trials each were carried out 2 and 4 hoursafter the treatment of the animals with the substance to be tested.

In the following table the results of the known peptide 4-10 ACTH arecompared with some peptides according to this invention.

Estimated Dosage potency in grn. Second Third ratio per First sessionsession compared I animal session, after after with 4-10 Peptide (s.e.)0 hour 2 hrs. 4 hrs. ACTH (i1) H-Met-GlwD-His-Phe-Arg-Trp-Gly-OH (440ACTH) 1% 8 8 7 1 H-Mct- Glu-D-His-Phe-O'H' 10 9 9 6 3H-Met-D-Glu-His-OH- 30 8 8 8 1-3 H-Met-Glu-D-His-PPA 10 9 7 5 3H-Met-Glu-D-His-Phe-Lys-Phe-OH 1; g g 6 10 TABLECntinued EstimatedDosage potency in gm. Second Third ratio per First session sessioncompared animal session, after after with 4-10 Peptide (5.0.) 0 hour 2hrs. 4 hrs. AOTH(:b1)

H-Met-Glu-D-His-Phe-Lys-Tra 1 9 9 7 50 H-Met-D-Glu-His-Phe-Lys-Phe-OH 1g1g 2 2 H-Met-Glu-D-His-Phe-LysPPA 10 9 8 8 10 H-Met(0)-Glu-D-His-Phe-Lys-Phe-OH 0. 3 9 7 6 100 H-fl-Ala-Glu-D-His-Phe-Lys-Phe-OH 1 9 9 7 50 H-Met-Glu-D-His-Phe-Lys-Trp-OH 10 99 6 3 H-Met-D-Glu-His-Phe-Lys-Trp-OH 30 8 8 7 13 With regard to thevarious abbreviations used throughout the specification and examples,the following remarks are made:

(I) If no optical configuration has been stated the L- form is meant.

(II) The following abbreviations have been used for the protecting oractivating groups:

Z:benzyloxy-carbonyl Boc:tertiary butyloxy-carbonyl ONB nitrobenzyloxytBu:tertiary butyl Me=methyl ONP:p-nitrophenyloxy Su:succinimido (III)For the solvents or reagents the following abbreviations have been used:

Bz:benzene To:toluene EtOH:ethano1 Bu:butanol Py=pyridine Ac orHac:acetic acid Fo=formic acid Am:arnyl alcohol iPro:isopropanolDMF:dimethylformamide THF :tetrahydrofuran DCCI:dicyclohexylcarbodiimideDCHU:dicyclohexyl-ureum TAA:triethylamine TFA:trifiuoroacetic acidWa:water (IV) For the amino acid the following abbreviations have beenused:

Met:methionyl Met O) :methionylsulfoxide (rac.) Met (d: O) :methionyl dsulfoxide Met l: O) :methionyl(l)su1foxide Met 0 :methionylsulfone GluQ) or Glu glutamyl (Q :OH) Glu (Q) or Gln:glutaminyl (Q:NH His=histidylPhe:phenylalanyl Arg: arginyl Lys:lysyl Gly: glycyl Val:valy1 Ala:alanyl (QC-M6) Ala: a-methylalanyl (V) Abbreviations used for otherresidues:

Amf: (N- l-phenylisopropyl) amino group (derived from amphetamine) Tra:(N-fl-indolylethyl) amino group (derived from tryptamine) PEA:(N-phenylethyl) amino group PPA: (N-phenylpropyl amino group HPEA:(N-p.hydroxy-phenylethyl amino group Desamino-Met:desamino-methionyl (or-methylthiobutyryl) Desamino-Met O) :desamino-methionyl sulfoxideDesamino-Met O :desamino-methionyl sulfone.

PREPARATION STARTING SUBSTANCES (A) Synthesis Boc-Met-D-Glu(OtBu)-His NH (1) Z-D-Glu(OtBu)-His-OMe:

Z-D-Glu(OtBu)-OH (6.62 g.) is dissolved in 30 ml. of acetonitril. Thissolution is cooled down to 0 C., after which 5.32 g. of H-His-OMe 2 HClin 30 ml. of cooled acetonitril and 2 equiv. TAA are added, after whichat 0 C. 4.62 g. of DCCI in ml. of acetonitril are added. The mixture isstirred for 2 hours at 0 C. and left to stand for hours at 0 C., afterwhich the resulting precipitate is filtered off and the filtrateevaporated in vacuum.

The residue is dissolved in ethyl acetate, washed with sodiumbicarbonate and saliferous water and dried. The solvent is distilled offin vacuum. The resulting oil is chromatographed over SiO (solventmixture BzzEtOH :82).

Yield: 3.5 g. of oil (foam). R1 in Bz:EtOH (8:2):0.57 on SiO (2)H-D-Glu(OtBu)-His-OMe.2 HCl:

Of the oil obtained above 2.9 g. are dissolved in ml. of methanol. Afterthe addition of 3.25 ml. of 4 N HCl and 600 mg. of Pd/C 10%, hydrogen isbubbled through the mixture. After 2.5 hours the mixture is filtered andthe filtrate evaporated.

Yield: 2.35 g. R1 in Am:Fo:Wa (7:2:1):0.33 on SiO (3) Boc-Met-D-Glu(OtBu) -His-OMe:

Boc-MetN H 1.59 g.) is dissolved in 15 ml. of DMF. This solution iscooled down to 0 C., after which 2.6 ml. of 4.85 N HCl/THF are added andat 20 C. 0.82 ml. of isoamyl nitrite. The mixture is stirred for 5minutes at 20 C., after which the azide which has formed meanwhile, isadded to a solution of 2.35 g. of H-D-Glu(OtBu)- His-OMe.2 HCl (A2) in20 ml. of DMF.

Then TAA is added to obtain pH 7.2.

The reaction mixture is stirred for 70 hours, after which it is filteredand the filtrate evaporated to dryness. The residue is dissolved inaqueous ethyl acetate and washed with water. The organic phase is dried,after which the ethyl acetate is evaporated to 15 ml. and diluted withm1. of petroleum ether. After one day the clear solution is evaporatedto dryness.

Yield: 2.63 g. of oil (foam). Rf in Bu:Ac:Wa (4:1:1) =0.76 on SiO (4)Boc-Met-D-Glu(OtBu)-His-N H 2.6 g. of ester (A3) are dissolved in 50 ml.of methanol, after which 2.6 ml. of hydrazine hydrate are added.

The reaction mixture is stirred for 5 hours, after which it isevaporated and set aside under ether at C.

After the mixture has been left to stand for 24 hours, the resultingprecipitate is filtered off.

Yield: 2.1 g. Rf in Am:iPro:Wa (10:4:5):0.36 on SiO (B) In the same wayas described in A are prepared:

(C) Synthesis Boc-Met-Glu(OtBu)-D-His-N H (1) Z-Glu (OtBu)-D-His-OMe:

Z-Glu(OtBu)-OH (66.2 g.) is dissolved in 115 ml. of acetonitril. Afterthe addition of 50 g. of H-D-His-OMel HCl in 115 ml. of acetonitrile,the solution is cooled to 0 C., after which 58 ml. of TAA are added andat 0 C. 38.8 g. of DCCI.

The mixture is stirred for 22 hours at 0 C., after Which it is filteredand the filtrate evaporated in vacuum, after which the residue is takenup in ethyl acetate. The organic phase is washed with a sodiumbicarbonate solution in water and water. After the addition of 575 ml.of ether and 115 ml. of petroleum ether the mixture is extracted 4 timeswith 100 ml. of 1 N HCl. After neutralizing the acid water extract with1 N sodium hydroxide (final pH 8.5) the mixture is extracted again withethyl acetate.

The organic layer is washed 3 times with saliferous water, dried andevaporated to 400 ml. After the addition of 575 ml. of ether and 116 ml.of petroleum ether, the dipeptide crystallizes out.

Melting point 73-76 C. R in AmzPyzWa (5:3:2): 0.60 on SiO 47.7 g. of theester from (1), dissolved in 336 m1. of methanol, are converted into thehydrazide by adding 12.5 ml. of hydrazine hydrate, after which themixture is stirred at 0 C. for 48 hours. Then the mixture is evaporatedto 100 ml., after which 475 ml. of distilled cold water are added. Afterfiltration the above peptide is obtained.

Melting point: 178-181 C.

(3) H-Glu(OtBu)-D-His-OMe.2 HCl:

Hydrogenation of 5.8 g. of dipeptide (C.l) as described in A2, yieldsthe dipeptide H-Glu(OtBu)-D-His- OMe.2HCl in 94% yield.

Rf in Am:Fo:Wa (712:1):032 on SiO (4) Boc-Met-Glu(OtBu)-D-His-OMe:

Boc-Met-N H (1.59 g.) is converted into the azide as described in A.3,and coupled to 2.35 g. of H-Glu(OtBu)- D-His-OMe.2HCl (C3).

The reaction mixture is stirred for 86 hours, after which it isevaporated to dryness.

The residue is taken up in aqueous ethyl acetate and washed 4 times withwater. The residue is dried, after which the ethyl acetate is evaporatedto dryness. The oily residue weighs 2.4 g.

Rf in Bu:Ac:Wa (4: 1:1)=0.74 on SiO The tripeptide (0.4) is convertedinto the hydrazide in the manner described before. The hydrazideobtained is dissolved in methanol and poured out into the tenfoldquantity of water.

Yield: 1.3 g. Rf in Am:iPro:Wa (10:4:5)=0.35 on SiO (D) OtherD-His-peptides (1) Boc-A-Glu-OtBu)-D-His-N I-I (A=,B-Ala,desamino-Met,Gly,(e-Me)Ala, Val or Ala):

(a) Boc-B-Ala-Glu(OtBu)-D-His-OMe:

Boc-B-Ala-N H (0.61 g.) is dissolved in 10 ml. of DMF, after which thesolution is cooled to 0 and 1.3 ml. of 4.85 N HCl/THF are added. Themixture is cooled down to 21, after which 0.41 ml. of isoamyl nitrite isadded. The mixture is stirred for 7 minutes, after which the azide hasformed. The azide solution is added to a pre-cooled solution of 1.2 g.of I-I-Glu(OtBu)-D-His- OMe.2 HCl in 10 ml. of DMF.

The reaction mixture is adjusted with TAA to pH 7.2, stirred for 86hours at 0 and evaporated to dryness. The oily residue ischromatographed over SiO Yield: 0.9 g. R7 in BuzAczWa (4:1:y) =0.73 onS102.

Conversion of 0.75 g. of ester into hydrazide is performed in the sameway as described in A.4.

Yield: 0.7 g. Rf in Am:iPro:Wa (10:4:5)=0.29 on SiO (c) In the same wayare prepared:

Desamino-Met-Glu(OtBu)-D-His-N H Boc-Gly-Glu (OtBu)-D-His-N HBoc-(u-Me)Ala-Glu(OtBu) -DHis-N H Boc-Val-Glu(OtBu)-D-His-N HBoc-Ala-Glu (OtBu -D-His-N H In the same manner as described in C theamino acid derivative Boc-Met-N H is coupled to H-Gln-D-His- OMe,obtained from the corresponding Z-protected peptide by hydrogenationwith 10% palladium on charcoal, yielding the protected peptide ester:Boc-Met-Gln-D-His- OMe, which peptide is immediately processed into thecorresponding hydrazide.

R in AmziProzWa (10:4:5)=0.27 on SiO (E) H-Phe-D-Arg-Oh (1Z-Phe-D-Arg(NO -OMe:

A solution of 12.8 g. of HD-Arg(NO )-OMe.HC1 in 200 ml. of DMF is cooleddown to +5 C., after which 4.8 g. of TAA are added. The resultingtriethylamine.HCl is filtered off, after which 20 g. of Z-Phe-ONP areadded. The mixture is stirred for 4 days at room temperature, afterwhich part of the DMF is distilled off and the residue diluted with 400ml. of ethyl acetate/water. The organic phase is washed with citricacid, ammonium-hydroxide and water, after which the ethyl acetate layeris dried and evaporated.

After recrystallization from ethyl acetate/petroleumether the meltingpoint obtained is 89-93 C. R in BzzEtOH (8:2)=0.62 on SiO,,.

(2) Z-Phe-D-Arg(NO )-OH:

Of the above dipeptide 5 g. are dissolved in dioxane and then saponifiedwith 1.1 equiv. sodium hydroxide. After 2 hours stirring, this solutionis acidified with 1 N hydrochloric acid (pH=2) and diluted with atenfold quantity of water. The resulting precipitate is stirred for 3hours at 0 and filtered off.

Yield: 3.1 g. Melting point=118121 C. (dec.).

Rf in BzzEtOH (8:2)=0.l1 on SiO (3 H-Phe-D-Arg-OH.acetate:

One gram of the Z-Phe-D-Arg(NO- )-OH (from 2) is dissolved in 20 ml. of90% acetic acid, after which mg. of palladium 10% on carbon are added.After hydrogen has been bubbled through, the black reaction mixture isfiltered over hyflo, the filtrate evaporated to dryness and the residuedissolved in t-butanol/water (1:1) and lyophilized.

Yield: 0.4 g. of dipeptide acetate.

R in BuzPyzAczWa (4:3/4:1/4:1) =0.21 on SiO 1 1 (4) In the same asdescribed in (3) the Z-Phe-D- Arg(NO )-OMe (from (1) is converted intothe H-Phe-D-Arg-OMe.acetate. R in Bu:Py:Ac:Wa (4:3/4:1/4:1)=0.40 on SiO(F) H-Phe-Lys(Boc)-OtBu Coupling of 4.2 g. of Z-Phe-ONP with H-Lys(Boc)-OtBu in the way described in E1 gives the dipeptide Z- Phe-Lys(Boc)-OtBuas a viscous oil.

R in Bz:EtOH (9:1)=0.57 on SiO Yield: 79%.

The dipeptide H-Phe-Lys(Boc)-OtBu is obtained in 95% yield byhydrogenating the peptide Z-Phe-Lys(Boc)- OtBu in methanol with 10%palladium on charcoal.

R) in BuzAczWa (4: 1:5)=0.41 on SiO (G) H-Phe-D-Lys(Boc)-OH (1Z-Phe-D-Lys(Boc) -OBZ1:

Starting from 17.7 g. of H-D-Lys(Boc)-OBzl.HCl the dipeptideZ-Phe-D-Lys(Boc)-OBzl is prepared in the same way as described in E1.

Yield after evaporation of the ethyl acetate: 72% (oil).

Rf in Bz:EtOH (9: 1)=0.54 on SiO (2) H-Phe-D-Lys(Boc)-OH:

Two grams of Z-Phe-Lys(Boc)-OBzl are dissolved in 50 ml. of methanol,after which 0.4 g. of palladium 10% on charcoal is added and hydrogen isbubbled through the mixture. After 5 hours filtration takes place, andthe filtrate is evaporated to dryness.

Rf in BuzPyzAczWa (4:3/4:1/4:1)=0.31 on SiO (H) H-Phe-N- (CH -N-BocCondensation of Z-Phe-ONP and Melting point: 131-133 C. Afterhydrogenation in methanol the is obtained. Rfi in BuzAczWa (4:1:5)=0.23.

(K) H-Phe-Lys(Boc)-Trp-OH and derivatives Z-Phe-ONP (4.6 g.) isdissolved in 60 ml. of DMF. H-Lys(Boc)-Trp-OMe (4.4 g.) is added, afterwhich the mixture is stirred at C. for 20 hours. Then 0.2 g. of2-dimethylamino-ethylamine is added. The reaction mixture is stirred foranother 2 hours and then evaporated. The residue is dissolved in 250 ml.of ethylacetate/water. This solution is successively washed with asolution of 5% potassium carbonate in water, 0.5 N hydrochloric acid andwater, after which the organic phase is dried on sodium sulphate.

After filtration the filtrate is evaporated to dryness and the oilyresidue saponified at once in dioxane, For this purpose 6.54 g. of esterare dissolved in 100 ml. of dioxane, after which 11.8 ml. of 1.5 N NaOHare added. After 2 hours stirring the resulting reaction mixture isacidified with 1.5 N hydrochloric acid to pH 7, after which the dioxaneis distilled off in vacuum.

To the residue ethylacetate/Water is added and the water layer adjustedto pH 2 with dilute hydrochloric acid, without the layers beingseparated. The organic phase is washed with water and then dried. Theethyl acetate is distilled ofi in vacuum to obtain an oil, which becomessolid after being stirred in ether.

R7 in Am:Py:Wa (5:3:2)=0.82 on SiO (2) H-Phe-Lys(Boc)-Trp-OH.HC1:

Of the tripeptide acid from (1) 1.85 g. are dissolved in ml. of DMF and1 equiv. hydrochloric acid. Palladium 10% on charcoal (350 mg.) isadded, after which hydrogen is bubbled through the mixture for 6 hours.After the reduction the catalyst is filtered oif and the filtrateimmediately processed further.

Rf in Am:Py:Wa (5:3 :2)=0.72 on SiO Two grams of the crude ester (see 1)are added to 25 ml. of a 10 N ammonia solution or dimethylamine solutionin methanol. The mixture is stirred for 2 days and after that it isevaporated to dryness and the residue recrystallized from methanol.

Rf in Bz:EtOH (8:2) =0.35 on SiO for the amide and 0.43 for thedimethylamide.

(4) Z-Phe-Lys(Boc)-Trp-OC H Z-Phe-Lys(Boc)-Trp-OH (from 1), 3.6 g., aredissolved in 45 ml. of DMF. Then 1.35 g. of undecyl bro mide and 1.0 g.of dicyclohexylamine are added. The mixture is stirred at 35 for 48hours and at 0 C. for 2 hours. The dicyclohexylarnmonium bromide formedis filtered off and the filtrate evaporated to dryness in vacuum. Theresidue is taken up in ethyl acetate, washed with 0.1 N HCl, water, a10% sodium bicarbonate solution and water. The organic phase is driedfor a short time on sodium sulphate, then partly evaporated in vacuumand crystallized by adding n.hexane.

R in Bz:EtOH (8:2)=0.48 on SiO (5) In the same day as described in K.2one gram of the tripeptides prepared in (3) and (4) is hydrogenated:

In this way are obtained the hydrochlorides of (L) H-Phe-Arg-Trp-OH (1)Boc-Arg(NO )-Trp-ONB:

Boc-Arg(NO )-OH (5.76 g.) is dissolved in 70 ml. of DMF and 2.52 ml. oftriethylamine. This solution is cooled down to 10 C., after which 2.38ml. of isobutylchloroformate are added and the mixture is stirred at 10C. for 10 minutes. To this solution a solution of 6.14 g. ofH-Trp-ONB.HC1 inv 40 ml. of cooled DMF and 3.01 ml. of triethylamine isadded. The mixture is stirred for 30 minutes at 10, for 3 hours at 0 andfor 20 hours at 20 C., after which the solvent is evaporated in vacuumand the residue taken up in ethyl acetate/water (10:1). The organicphase is washed with water, 5% sodium bicarbonate and water, dried andevaporated to dryness in vacuum.

Yield: 8.5 g. of oil. Rf in Bz:EtOH (8:2)=0.79 on SiO (2) H-Arg(NO)-Trp-ONB:

One gram of the peptide prepared in 1) is dissolved in 20 ml. ofmethylene chloride, after which hydrogen chloride gas is bubbled throughthe solution while cooling. After 1 hours stirring the precipitate isfiltered oil and thoroughly washed with dry methylene chloride. The precipitate is immediately processed further.

R in Am:Py:Wa (5:3:2)=0.66 on SiO (Rf starting substance 0.93).

(3 Z-Phe-Arg(NO -Trp-ONB:

Three grams of the dipeptide from (2) are dissolved in 20 ml. of DMF.After cooling this solution to 0 C., 1.12 ml. of TAA and 2.12 g. ofZ-Phe-ONP are added. The reaction mixture is stirred at 0 C. for 2 hoursand at 20 C. for 20 hours, and then evaporated in vacuum. The oilyresidue is dissolved in ethyl acetate/water (10:1) and processed asdescribed in (1). The organic phase is evaporated and the residuedissolvedS ml. of methanol and gently added to ml. of dry ether toprecipitate the peptide.

Yield: 2.9 g. R7 in Bz:EtOH (8:2)=0.48 on SiO;,,.

(4) H-Phe-Arg-Trp-OH:

Two grams of the above-mentioned peptide from (3) are dissolved in 40ml. of acetic acid, after which 400 mg.

of 10% palladium on charcoal are added and the mixture is hydrogenatedfor 2 days (Parr).

After filtration the acetic acid is evaporated in vacuum, after whichthe residue is stirred into dry ether. Yield: 98%. The reddish foam isdried over solid potassium hydroxide. The substance contains 1.1molecule of acetic acid.

R) in AmsPyzWa (5:3:2)=0.17 on SiO (M) H-Phe-Arg-Tra (l) Boc-Arg(NO)-Tra:

Boc-Arg(NO )-O-H (5.76 g.) is dissolved in 70 ml. of DMF and 2.52 ml. ofTAA. This solution is cooled down to 10 C., after which 2.38 ml. ofisobutylchloroformate are added and the mixture is stirred at 10 C. foranother 10 minutes. To this solution a solution of 2.9 g. of tryptaminein 10ml. of DMF are added, the temperature being maintained at about 10C. The mixture is stirred for 30 minutes at 10 C., for 2 hours at andfor 18 hours at 20, after which the solvent is evaporated off in vacuumand the residue taken up in ethyl acetate/ water. The organic phase iswashed with water, bicarbonate and water, after which it is dried andevaporated.

Yield: 7.4 g. oil. Rf in Bz2EtOH (8:2):054 on SiO (2) Z-Phe-Arg(NO )-Traand H-Phe-Arg-Tra:

Z-Phe-ONP (2.1 g.) is dissolved in ml. of DMF and 10 ml. of ethylacetate. Then 1.81 g. of H-Arg (-NO )-Tra (obtained by deprotection ofthe amide from (1)) are added, after which the mixture is stirred for 30minutes at 10 C. and for hours at 20 C.

The reaction mixture is processed as described in L3. Yield: 1.3 g.;melting point 13 1133 C. Rf in BztEtOH (8:2)=0.47 on SiO Of the compoundobtained 1.3 g. are converted into H-Phe-Arg-Tra by means of 10%palladium on charcoal and hydrogen (Parr). Yield: 72%.

(N) H-Phe-Lys(Boc)-Tra (1 Z-Phe-Lys (Boc)-Tra:

Starting from 3.4 g. of Z-Phe-ONP and 3 g. of H-Lys (Boc)-Tra (preparedfrom Z-Lys( Boc)-Tra; melting point 77-80 C.) Z-Phe-Lys(Boc)-Tra isobtained in the way described in E1.

Yield: 65%. Melting point: BzzEtOI-I (8:2)=0.7O on SiO (2)H-Phe-Lys(Boc)-Tra:

Two grams of the peptide from 1) are dissolved in ml. of methanol andhydrogenated with hydrogen in the presence of 10% palladium on charcoal,in a conventional manner. After evaporation to dryness a foam isobtained. Yield: 95%.

R) in BucAczWa (4:l:1)=0.80 on SiO (O) H-Phe-Lys(Boc)-Phe derivatives 1)Z-Phe-Lys(Boc)-Phe-OMe:

H-Lys(Boc)-Phe-OMe (4.24 g.) is dissolved in 25 ml. ofdimethylformamide, after which 4.77 g. (11.4 mmol) of Z-Phe-ONP areadded to this solution. The mixture is stirred for about 24 hours, afterwhich the solvent is evaporated off in vacuum. The residue is dissolvedin a mixture of 120 ml. of ethyl acetate and ml. of water, after whichthe ethyl acetate phase is Washed successively with 0.1 N HCl, water, asodium carbonate solution (5%) and water. The ethyl acetate in distilledoff and the residue recrystallized from ethyl acetate, to which a littlepetroleum ether has been added.

R) is BzzEtOH (8:2) =0.76 on SiO (2) H-Phe-Lys(Boc)-Phe-OMe:

Of the peptide obtained in (1) 1.9 g. are dissolved in ml. of methanol.To this solution 0.5 g. of 10% palladium on carbon is added. Thenhydrogen gas is bubbled through the mixture, after which the catalyst isfiltered 0E and the filtrate evaporated to dryness.

R in AmzPyzWa (5:3:2)=0.35 on SiO 125-129 C. R in (3) H-Phe-Lys(Boc)-Phe-NH Of the ester obtained in 1) 500 mg. are dissolved in methanol,after which the solution is saturated with ammonia. The mixture isstirred for 24 hours. The amide crystallizes from the solution.

In the way described in (2) the protecting Z-group is removed from thisamide.

Rf in ArnzPyzWa (5:3:2)=0.23 on SiO (4) H-Phe-Lys(Boc)-Phe-OH:

Of the peptide-ester obtained in (2) 0.5 g. is dissolved in 6 ml. ofmethanol, after which 1 equiv. NaOH is added. The mixture is stirred forone hour, after which it is gently acidified to precipitate thetripeptide acid.

R7 in AmzPy2Wa (5:3:2) =0.18 on SiO (P) H-Phe-D-Lys(Boc)-Phe-OtBu (1Z-D-Lys (Boc) -Phe-OtBu:

Z-D-Lys(Boc)-ONP (10.03 g.) is dissolved in 50 ml. of DMF. This solutionis cooled down to '20 C. and added to a solution of 4.1 g. of H-Phe-OtBuin 75 ml. of DMF.

The reaction mixture is stirred for 1 hour at 0 C. and for 20 hours at20 C., after which it is evaporated to dryness. The yellow residue isdissolved in ethyl acetate/ water and washed with 5% potassiumcarbonate, water, 5% citric acid and water. The organic phase is driedand evaporated to dryness. The residue is dissolved in ethyl acetate,after which enough petroleum ether 40/60 is added to cause a turbidity.Then the resulting precipitate is filtered off.

R in BzzEtOH (9:1)-=0.64 on SiO (2) H-D-Lys(Boc)-Phe-OtBu:

Of the dipeptide 3 g. are dissolved in 60 ml. of methanol. Thenpalladium 10% on charcoal is added, after which hydrogen is bubbledthrough the mixture till no more CO escapes (2 hours). After filtrationover hyflo, the filtrate is evaporated to dryness to obtain a foam.

Rf in TozEtOH (9: 1)=0.21 on SiO 3) Z-PheD-Lys (Boc)-Phe-OtBu:

Z-Phe-ONP (2.18 g.) is dissolved in 15 ml. of DMF. Then a solution of2.24 g. of H-D-Lys(Boc)-Phe-OtBu from (2) in 30 ml. of DMF is added,after which the mixture is stirred for 15 hours at 20 C. Afterevaporation of the yellow solution, the residue is dissolved in 15 ml.of ethyl acetate, after which 50 ml. of petroleum ether are added. Thenthe mixture is left to stand at 0 C. for 8 hours, after which theprecipitate formed is filtered off. Melting point 135-138 C.

Rf in TozEtOH (9:1)=O.60 on SiO (4) H-Phe-D-Lys(Boc)-Phe-OtBu:

Of the tripeptide obtained in (3) 2.5 g. are hydrogenated in the sameway as described in (2).

Rf in TozEtOH (8:2)=0.43 on SiO (Q) H-Phe-Lys (Boc -phen.ylalkylamide 1)Z-Lys(Boc)-PPA:

Z-Lys(Boc)-ONP (10.33 g.; 20.6 mmol) is dissolved in ml. of methylenechloride at about 0 C. Then 2.7 g. of 3-phenylpropylamine are added tothis solution, after which the mixture is stirred at 0 C. for 1.5 hoursand then at room temperature for 18 hours. The solvent is evaporated andthe residue dissolved in 200 ml. of ethyl acetate. Then the ethylacetate solution is Washed successively with a 10% sodium carbonatesolution, a 30% NaCl solution, 0.1 N HCl and a 30% NaCl solution. Theethyl acetate layer is dried and evaporated to a volume of about 80 ml.After that enough ether is added to cause a turbidity, after which themixture is set aside in a refrigerator. After 2 hours the precipitateformed is filtered 01f. Melting point 7879 C.

Rf in BzzEtOH (9:1)=0.53 on SiO (2) H-Lys(Boc)-PPA:

Of the compound obtained in (1) 8.75 g. are dissolved in ml. of methanolto which 1.21 g. of 10% palladium on charcoal has been added. Whilestirring, hydrogen in 15 bubbled through the mixture for 3.5 hours,after which the catalyst is filtered off. The filtrate is evaporated todryness to obtain a practically colourless oil, which is directly usedfor further reactions.

R in Am:Fo:Wa (7:2:1)=0.58 on SiO 3) Z-Phe-Lys (Boc)-PEA:

Of the protected amino acid derivative obtained in (2) 6.39 g. aredissolved in 68 ml. of dimethyl formamide, after which a solution of7.61 g. of the ZPhe-ONP in 20 ml. of dimethyl formamide is added. Themixture is stirred at room temperature for 20 hours and then the solventis evaporated off in vacuum. The residue is dissolved in 170 ml. ofethyl acetate and washed successively with a 5% potassium carbonatesolution, a 30% NaCl solution, 0.1 N H01 and a 30% NaCl solution. Theethyl acetate layer is then dried on Na SO and evaporated to about 100ml. The solution is set aside in a refrigerator for 3 days, during whichperiod the peptide completely crystallizes. Melting point 134136 C.

Rf in BZ:EtOH (8:2)=0.72 on SiO (4) HPhe-Lys(Boc)-PPA:

Of the peptide derivative obtained in (3) 9.07 g. are dissolved in 300ml. of dimethyl formamide to which 4 ml. of 4 N HCl and 1.5 g. of 10%palladium/charcoal has been added. While stirring, hydrogen is bubbledthrough the mixture for 3.5 hours, after which the catalyst is filteredoil and the filtrate evaporated to dryness to obtain a practicallycolourless oil.

R in Bu:Ac:Wa (4:1:1)=0.63 on SiO (5) In the same way are prepared:

(1 H-Phe-Lys (Boc)-L-Amf Rf in Bu:Ac:Wa (4:1:1)=0.63 and SiO (2)H-Phe-Lys(Boc)-PEA R in Bu:Ac:Wa (421:1):065 on SiO (3 I-I-Phe-D-Lys(Boc -HPEA Rf in TozEtOH (8:2) =0.29 on SiO (R) I-I-Phe-Lys(Boc)Phe-Glyderivatives (1 Z-Phe-Lys (=Boc)-N H Z-Phe-Lys(Boc)-OMe (12.0 g.) isdissolved in 30 ml. of methanol and 6 ml. of hydrazine hydrate. Themixture is left to stand for 24 hours and then stirred at C. for 2hours. The resulting precipitate is filtered ofi, washed with coldmethanol and dried.

Of the above hydrazide 5.42 g. are dissolved in 20 ml. of DMF. Thesolution is cooled down to 0, after which 2 equiv. hydrochloric acid/THFare added and the mixture is cooled down to -20 C. Then 1.35 ml. ofisoamylnitrite are added, after which the mixture is stirred at -20 C.for 7 minutes. This mixture is added to a solution of 3.5 g. ofH-Phe-Gly-OBzl.HCl and 4.2 ml. of triethylamine (pH 7). The mixture isleft to stand at 0 C. for 70 hours, after which the solvent is distilledoif and the residue taken up in ethyl acetate/ water, after which theorganic phase is washed with 0.1 N HCl, sodium bicarbonate and water.The ethyl acetate layer is dried over sodium sulphate, after which theethyl acetate is distilled off in vacuum.

Rf in BZ:EtOH (8:2)=0.71 on SiO (3 H-Phe-Lys (Boc)-Phe-Gly-OH:

One gram of the peptide obtained in (2) is dissolved in methanolzwater1:1 (20 ml.). Then 10% palladium on charcoal is added, after whichhydrogen is bubbled through the mixture for 5 hours. After filtrationover Hyfio, the filtrate is evaporated and stirred into dry ether.

Rf in Bu:Ac:Wa (4: 1: l)=0.43 on SiO (S) SynthesisH-Phe-D-Lys(Boc)-Phe-Gly-OtBu (1 Z-D-Lys (Boc)- PheGly-OtBu:Z-D-Lys(Boc)-O'NP (10.03 g.) is coupled to a solution of 5.1 g. ofH-Phe-Gly-OtBu in ml. of DMF and then evaporated. The resulting residueis dissolved (in ethyl acetate) again in the way described, washed andevaporated to dryness. The residue is directly processed further.

Rf in To:EtOH (9: 1) =0.63 on SiO (2) H-D-Lys(Boc)-Phe-Gly-OtBu:

Hydrogenation gives the tripeptide in practically quantitative yield asa foam.

R) in To:EtOH (9:1)=0.24 on SiO Reaction of 2.18 g. of Z-PheONP with 2.6g. of HD- Lys ('Boc)-Phe-Gly-OtBu in 30 m1 of DMF, for 24 hours, givesthe tetrapeptide in 75% yield after crystallization from ethylacetate-petroleum ether. Before the crystallization the ethyl acetatelayer was washed with citric acid and a 5% sodium bicarbonate solution.

Melting point: 78-80 C. Rf in TozEtOH (8:2) =0.85 on SiO 4) H-Phe-D-Lys(Boc) -Phe-Gly-OtBu:

Hydrogenation of the tetrapeptide described in (3) givesH-Phe-D-Lys(Boc)-Phe-Gly-OtBu as a foam.

Rf in Bu:Py:Ac:Wa (42% :Mt:1)=0.57 on SiO EXAMPLE I H-Met-D-Glu-His-OH(1 'Boc-Met-D-Glu( OtBu)-His-OH:

rBoc-Met-D-Glu(OtBu)-His-OMe (2.34 g.; A3) is dissolved in 50 ml. of 50%dioxane. To this solution 25 ml. of 2 N sodium hydroxide are added,after which the mixture is stirred at 20 C. for 30 minutes. The reactionmixture is neutralized to pH 7, after which it is evaporated to drynessin vacuum and the residue taken up in ethyl acerate/Water. [After thatthe pH of the water layer is adjusted to 4 with 2 N hydrochloric acid.The water layer is evaporated to dryness and the residue stirred into 25ml. of abs. methanol, after which the precipitate is filtered off. Thefiltrate is evaporated to dryness.

(2) H-Met-D-Glu-His-OH:

One gram of tripeptide from (1) is dissolved in 10 ml. of trifluoroacetic acid and the mixture is stirred for 30 minutes. The reactionmixture is added dropwise to peroxide-free ether, after which theprecipitate is filtered OE and triturated with ether. The residue isdried over solid potassium hydroxide. After being dissolved in 25 ml. oft.butanol-water (1:1), Dowex X-8, in the acetate form is added toexchange the trifluoro acetic acid for acetic acid (final pH 55.4). Theion exchanger is filtered off and the filtrate lyophilized.

Rf in BuzPyzAczWa (4%: A:1)=0.13 on SiO EXAMPLE II H-A-D-Glu-His-(N-phenylalkyl) amides (A=Met, Desamino-Met, ,B-Ala or Val) 1)Boc-Met-D-Glu (OtBu) -His-PEA:

Of the tripeptide Boc-Met-D-Glu(OtBu)-His-N H from A.4 2.87 g. aredissolved in 20 ml. of DMF. The solution is cooled down to 0 C., afterwhich 5.94 ml. of 1.56 N HCl in THF and at 20 C. 0.42 ml. ofisoamylnitrite are added, after which the azide is formed. This azide isadded to 3.2 mmol phenylethylamine, after which the final pH is adjustedto 7.2 with TAA.

After 70 hours stirring at 0, the reaction mixture is evaporated invacuum and the residue taken up in ethyl acetate. The organic phase iswashed with water, a 5% sodium bicarbonate solution and water, and thendried and distilled off in vacuum.

Yield: 1.9 g. of oil, Rf in BZ:EtOH (8:2)=0.51 on SiO (2) In the sameway are prepared:

Boc-Met-D-Glu(OtBu)-His-Amf: Rf in BZ:EtOH

(8:2)=0.49 on SiO Boc-Met-D-Glu(OtBu)-His-PPA: R in BZ:EtOH

(8:2)=0.51 on SiO (3 H-Met-D-Glu-His- (N-phenylalkyl) amide:

In the Way described in Example 1.2 one gram of peptide from 11:1 or11.2 is treated with TFA and the trifiuoro-acetate obtained convertedinto the acetate.

Yield 70-75%.

The acetates are obtained of:

*Rf in Bu Py Ac Wa (4:3/4c1/4z1) on S102.

(4) 1n the way described in Example 111 some hydrazides prepared in Bare converted into the azide and coupled to phenylethylamine, afterwhich the protecting groups are removed with trifiuoro acetic acid(Example 11.3). Then the trifiuoro acetic acid is exchanged for aceticacid.

Obtained the acetates of:

H-fl-Ala'D-Glu-His-PEA: Rf*:0.34 H-Val-D-Glu-His-PEA: Rf*=0.37Desamino-Met-D-Glu-I-Iis-PEAr Rf*=0.44

*R in Bu Py Ac Wu (4 3/4 1/4 1) on S102.

EXAMPLE III H-A-Glu-D-HiS-Phe-OH, H-Met-Glu-D-His-PPA and derivativesthereof (1) Boc-Met-Glu(OtBu)-D-His-Phe-OtBu:

Boc-Met-Glu(OtBu)-D-His-N H (1.87 g.; C.5) is converted into the azideas described in Example 11.1.

The azide is added to a solution of 0.52 g. of H-Phe- OtBu in 10 ml. ofDMF cooled down to (final pH 7.2 by means of TAA) and the reactionmixture is stirred at 0 for 71 hours. After evaporation of the reactionmixture the residue is dissolved in aqueous ethyl acetate, after whichthe ethyl acetate is Washed twice with saliferous Water and then dried.The organic phase is evaporated and the residue dried.

Yield: 19 g. Rf in BzzEtOI-I (8:2):090 on SiO (2)H-Met-Glu-D-His-Phe-OH:

Of the tetrapeptide (111.1) 0.5 g. is treated in 20 ml. of 90% trifluoroacetic acid, as described in Example 1.2.

S ('1 \)*o:rn.Rj in Bu Py: Ac Wa (4:3/4:1/4:1):0.26 on (3) 1n the Waydescribed in 1 and 2 the following acetates are prepared starting fromBoc-Met-Glu(OtBu)-D- His-N H and H-Phe-oC l-l H-Phe-OMe orphenylpropylamine:

H-A-Glu-D-His-Phe-L(or D)-Lys-0H (A Met, fi-Ala or Desamino-Met) (1)Boc-Met-Glu( OtBBuB)-D-His-Phe-Lys (Boc)-OtBu:

Boc-Met-Glu(OtBu)-D-His-N H (C5), 1.87 g., are converted into the azidein the manner described in Example 11.1.

The azide solution is added to 3.2 mmol H-Phe-Lys (Boc)-OtBu CF) in ml.of DMF, after which the pH of the reaction mixture is adjusted to 7.2with TAA. After having been left to stand for 70 hours at 0 C., thereaction mixture is evaporated to dryness in vacuum and the 18 residuetaken up in aqueous ethyl acetate. The ethyl acetate is Washed 3 timeswith water, dried and distilled 011 in vacuum, after which an oilremains.

Rf in BzzEtOH (9:1)=0.30 on SiO If the azide prepared in Example IV.1 isadded to 3.2 mmol H-Phe-D-Lys(Boc)-OH (G2) and 0.45 ml. of TAA in 10 m1.of DMF 1.7 g. of oil is obtained after the mixture has been processed asdescribed above and extracted with saliferous water.

Rf in BzzEtOH (8:2)=0.21 on SiO (3) Removal protecting groups:

Of the peptides IV.1 0r IV.2 0.5 g. is treated in the way describedbefore. The product is exchanged for acetate after which the followingpeptides are obtained as an acetate.

H-Met-Glu-D-His-PheLysOH: Rf*=0.20 on SiO H-Met-Glu-D-His-Phe-D-Lys-OH:Rf*=0.18 on SiO (4) In the same way are prepared:

H-fi-Ala-Glu-D-His-Phe-Lys-OH by starting from the hydrazide prepared inD.1.b; Rf in Bu:Py:Ac:Wa (4:3/ 4:1/4:1)=0.16 on SiO andDesamino-Met-Glu-D-His- Phe-Lys-OH by starting from the hydrazideprepared in D.l.c.

Rf=0.28 on SiO EXAMPLE V H1Met-D-Glu-His-Phe-(Nam in'op entyl)-amideprepared in H, is coup-led to Boc-Met-D-Glu(OtBu)-His- N prepared inExample 11.1. After 70 hours stirring the condensation product isprocessed in the way described in Example 11.1 and the ethyl acetatelayer is added dropwise to petroleum ether to obtain a precipitate.

R in BurAczWa (4:1:5)=0.63 on SiO (2) Removal protecting groups:

In a conventional manner the tetrapeptide amide is treated with TFA andthe trifluoro acetate obtained converted to the acetate.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1)=0.25 on SiO EXAMPLE VIH-Met-D-Glu-His-Phe-D-Arg-OH and methylester 1) Boc-Met-D-Glu(OtBu)-His-Phe-D-Arg-OH:

Coupling of Boc-Met-D-GluODtBu)-His-N prepared in Example 11.1, andHPhe-D-Argo-OH.acetate (E3) in the way described in Example I1.1 givesthe above pentapeptide as a amorphous substance in 62% yield.

Rf in Bz:E'tOH (8:2)=0.16 on 810;, and in (2) In the same way the azidesBoc-Met-D-Glu- (Ot Bu)-His-N and Desamino-Miet-D-Glu(OtBu)-His-N arecondensed with H-Phe-D-Arg-OMe (15.4)

(3) Removal protecting groups:

0.5 g. of peptide from 1 (or 2) is dissolved in 15 ml. of trifiuoroacetic acid/water (9:1) and after that the mixture is stirred for 30minutes. The resulting TFA salt is converted into the acetate and storedover solid potassium hydroxide (see Example 1.1).

Obtained the acetate of:

*Rf in BuzPy2AczWa (4:3/4:1/4:1) 011 S102.

1 9 EXAMPLE v11 H1Met-D-Glu-His-PheLys-Trp-OH and derivatives thereof 1)Boc-Met-D-Glu(OtBu)-His Phe Lys(Boc) Trp- OH:

Of the tripeptide hydrazide: Boc-Met-D-Glu(OtBu)- His-N H (A.4) 1.87 g.are dissolved in 20 ml. of DMF. The solution is cooled down to and afterthat 5.94 ml. of 1.566 N HCl/THF are added and at 20 C. 0.42 ml. ofisoamylnitrite, by which the azide is tor-med. This azide is added to asolution of 1.45 g. of H-Phe- Lys(-Boc)-Trp-OH.HC1 (K.2) in 15 ml. ofDMF, after which the pH is adjusted to 7.2 with TAA.

After 70 hours stirring at 0 C., the reaction mixture is evaporated andthe residue taken up in aqueous ethyl acetate and washed with saliferouswater. The organic layer is dried and evaporated. Then petroleum etheris added to obtain a solid substance.

Yield: 1.66 g.

Rf in BuzPyzAczWa (4:3/4:1/4:1)=0.85 on Si0 (2)H-Met-D-Glu-His-Phe-Lys-Trp-OH:

Of the above peptide 1.6 g. are dissolved in 25 ml. of 90% trifluoroacetic acid. The light red solution is left to stand for 30 minutes,after which it is poured into 400 m1. of dry, peroxide free ether. Thesolid substance is filtered off and dried. The salt obtained isdissolved in 70 ml. of t-BuOH:water=1:1. After that the mixture isstirred with Dowex X-8 in the acetate form to obtain a final pH of -5.5.The mixture is stirred for 1 hour. After filtration the filtrate is'lyophilized to obtain the acetate of H-Met-DGlu-His-Phe-Lys-Trp-OH.

Rf in Bu:Py:Ac:Wa '(2:3/4:1/4:1)=0.17 on SiO (3) In the same way asdescribed in (1) and (2) the =azide described in (1) is coupled toH-Phe-Lys(Boc)- Trp-N=H or H-Phe-Lys(Boc)-Trp=OC H prepared in KS, andthen the protecting groups are removed.

Obtained the acetates of:

H-Met-D-Glu-His-Phe Lys-Trp-NH and H-Met-D-Glu-His-Phe-Lys-Trp-OC HEXAMPLE VIII 7 H-A-Glu D-His-Phe-Lys-Trp-OH 1) Z-Glu (OtBu)-D-His-Phe-Lys (Boc) Trp-OH:

Z-Glu(Ot-Bu)-D-His-N H (4.1 g.; O2) is dissolved in 60 ml. of DMF. Thesolution is cooled down to 0 C., after which 5.5 ml. of 1 N THF/HCl isadded and at 20 C. 1.35 ml. of isoamylnitrite. The azide formed duringminutes stirring at 20 C. is added to a solution of 8.4 mmolH-Phe-Lys(Boc)-Trp-OH.HC1 (K.2) in 20 ml. of DMF, after which the pH ofthe reaction mixture is adjusted to 7.2. The mixture is stirred for 100hours at 0 C., after which it is filtered off and the filtrateevaporated to dryness in vacuum. The residue, dissolved in ethylacetate, is washed successively with water, a 10% citric acid solution,water and saliferous water and then dried. The ethyl acetate isdistilled off and after that the residue is taken up in DMF (25 ml.) andadded dropwise to 600 ml. of ethyl acetate/ether 1:2). Yield 5.3 g.

(2) H Glu'(Ot Bu) D His Phe Lys(Boc) Trp- OH.HC1:

Five grams of the above peptide 1. are dissolved in 100 ml. of DMF and2.2 ml. of 4 N HCl. After the addition of 1 g. of palladium 10% oncharcoal hydrogen is hub bled through the mixture. After 24 hours themixture is filtered and the filtrate evaporated in vacuum. The residueis recrystallized from methanol-ether. Yield: 4.5 g.

Rf in AmzFozWa (7:2: 1)=0.68 on "SiO (3) Boc-Met-Glu(OtBu)-D-His-Phe-Lys (Boc) Trp- OH:

Boc-Met-N H (0.982 g.; 3.73 mmol) is dissolved in 20 ml. of DMF. Thesolution is 9991ed to after which 20 3.8 m1. of 2 N HCl/THF are addedand at 20 C. 0.5 m1. of isoamylnitrite, after which the mixture isstirred at 20 C. for 10 minutes.

The azide is added to 3.2 g. of H-Glu(OtBu)-D-HisPhe-Lys(Boc)-Trp-OH.HC1 (Example VIII.2) after which the pH is adjustedto 7.1 with TAA.

The mixture is stirred at 0 C. for 71.5 hours, after which the filtrateis evaporated to dryness and the residue stirred into 100 ml. of ethylacetate. Yield: 3.05 g.

(4) By coupling the required Boo-amino acid-azide toH-Glu(OtBu)D-His-Phe-Lys(Boc)-Trp-OH.HC1 in the same manner as describedin 3, the following peptides are prepared:

Boc-Val-Glu OtBu D-His-Phe-Lys Boc) Trp-OH Boc-( u-Me) Ala-Glu (OtBu)D-His-Phe-Lys (Boc) Trp-OH Boc-Gly-Glu (OtBu) D-His-Phe-Lys (Boc Trp-OHDesarnino-Met-Glu OtBu D-His-Phe-Lys Boc Trp-OH (5)H-A-Glu-D-His-Phe-Lys-Trp-OH:

Two grams of a peptide described in Example VIII.3 or VIII.4 aredissolved in 45 ml. of 90% trifluoro acetic acid and treated asdescribed before.

By exchanging the trifiuoro-acetate for acetate With Dowex X-8 thehexapeptide is obtained as an acetate in 92% yield.

*Rf in Bu Py AC Wa (2 33/4: 1/4 1) on SiO2.

EXAMPLE IX H-Met-Glu-D-His-Phe-Lys-Trp-OH and derivatives (1)Boc-Met-Glu(OtBu D-I-lis-Phe-Lys (Boc)-Trp-Y:

Boc-Met-Glu(OtBu)-D-His-N H (9.35 g.) is converted in a conventionalmanner into the azide (Example III.1), after which the solvent is addedto bring the volume of the solution at exactly 50 ml.

Of this solution /5 part is added to: 3.2 mmol H-Phe-Lys(Boc)-Trp-OH.HC1 (K2) in 10 ml. of DMF at 0 C.;

1', part of the solution is added to: 3.2 mmol H-Phe-Lys Boc)-Trp-NH.HCl (K.5) in 10 ml. of DMF at 0 C /5 part to: 3.2 mmol ofH-Phe-Lys(Boc) Trp- (K.5) in 10 ml. of DMF at 0 C., and

A; part to: 3.2 mmol of H-Phe-Lys Boc) Trp-N CH .HC1

in 10 ml. of DMF at 0 C., after which the pH of each of the mixtures isadjusted to 7.2-7.3 with TAA. The mixtures are stirred at 70 hours at 0C. after which each reaction mixture is evaporated in vacuum (bath 40C.) and the residue taken up in aqueous ethyl acetate. This layer iswashed 3 times in water, after which the ethyl acetate is evaporated offin vacuum, the residue taken up in alcohol and the solution diluted withperoxide-free ether to obtain a precipitate.

Yield:

71% hexapetitde acid; Rf in BzzEtOH (8:2)=0.25 on SiO 57% hexapeptideamide; Rf in Bz:EtOI-I (8:2)=0.38 on SiO 2 61% hexapeptide ester; Rf inBzzEtOH (8:2):0t49 on SiO 50% hexapeptide dimethylamide; Rf

(8:2) =0.45 on SiO (2) Removal protecting groups: Of the hexapeptideprepared above 0.5 g. is dissolved in BzzEtOH in 25 ml. of 90% trifiuoroacetic acid and stirred at 20 C. for 1 hour. The mixture is poured into300 ml. of dry peroxide-free ether, after which the precipitate isfiltered and dried over solid KOH. 2 50 mg. of substance are dissolvedin 30 ml. of t-butanolzwater (1 1) and stirred with Dowex X8 in theacetate form to obtain a final pH of 5.2.

After filtration 20 ml. of t-butanol-water are added and the entirefiltrate is evaporated to dryness.

Obtained in this way the acetates of H-Met-Glu-D-His-Phe-Lys-Trp-OH:Rf*=0.32 on SiO H-Met-Glu-D-His-'Phe-Lys-Trp-NH Rf*=0.37 on SiOH-Met-Glu-D-His-Phe-Lys-Trp-OC H Rf*=0.47 on SiOH-Met-Glu-D-His-Phe-Lys-Trp-N(CH Rf*=0.42 on SiO *Rf in Bu:PyAc:Wa(2:3/z1/4: EXAMPLE X H-Met-Glu-D-His-Phe-Arg-Trp-OH Starting fromBoc-Met-Glu(OtBu)-D-His-N prepared in accordance with Example 111.1, andH-P'he-Arg-Trp-OH from L4 the hexapeptide is obtained after 70 hoursstirring at C.

The residue obtained after evaporation of the reaction mixture in vacuumis taken up in DMF and poured into the tenfold quantity of water. Theresulting precipitate is dried and treated in the manner describedbefore.

The amorphous product, which has a light colour, is purified further viaa counter current distribution; system: Bu:Ac:Wa (4: 1

EXAMPLE XI M-Mct-D-Glu-His-Phe-Arg-Tra, H-Met-D-Glu- His-Phe-Lys-Phe-OH(1) Starting from 1.87 g. of Boc-Met-D-G-lu(OtBu)- His-N H and 3.2 mmolH-Phe-Arg-Tra (M2) the pentapeptide amide is prepared by the processdescribed in Example II.1. The reaction mixture is left to stand for 84hours at 0 C., evaporated to dryness and taken up in aqueous ethylacetate.

The mixture is extracted 3 times with Water, after which the ethylacetate is dried and distilled off in vacuum. The residue is taken up inDMF and added dropwise to ethyl acetate/petroleum ether (1:1) to obtaina precipitate. Yield: 1.72 g. Rf in BuzPyzAczWa (4:3/4:1/4:l) =0.51 onSiO Treatment as described before with trifluoro acetic acid andexchanging this product against acetic acid gives the acetate ofH-Met-D-Glu-His-Phe-Arg-Tra in 82% yield.

(2) In the same manner as described in (1) the peptideH-Met-D-Glu-His-Phe-Lys-Phe-Oh.acetate is prepared, starting fromBoc-Met-D-Glu(OtBu)-His-N H and H-Phe-Lys(Boc)-Phe-OH (0.4).

Rf in Bu:Py:Ac:Wa (4:3/4:l/4:1)=0.l9 on SiO EXAMPLE XIIH-A-Glu-D-His-Phe-LysTra (1 Boc-Met-Glu( OtBu)-D-His-Phe-Lys(Boc) -Tra:

Boc-Met-Glu(OtBu)-D-His-N H (1.87 g.) is dissolved in 20 ml. of DMF,converted into the azide in accordance with Example "111.1 and coupledto the H-Phe-Lys- (Boc)-Tra prepared in N2. The pH is adjusted to 7.2with TAA, after which the reaction mixture is stirred at 0 C. for 70hours and then filtered. The DMF is distilled off in vacuum and theresidue is taken up in ethyl acetate, washed with water, a 5% sodiumbicarbonate solution and water, after which the ethyl acetate layer isdried. This oragnic phase is distilled off, after which the residue istaken up in alcohol and added dropwise to peroxide-free ether. Theprecipitate formed is filtered otf.

Melting point: 203 dec.

(2) Removal protecting groups:

0.5 g. of peptide from (1) is dissolved in 25 m1. of 90% trifluoroacetic acid and the mixture is stirred for 30 minutes. In the mannerdescribed before, the trifiuoro acetate is isolated, dried and exchangedfor the acetate, after which the acetate of H-Met-Glu-D-His-Phe-Lys-Trais obtained by lyophilizing the filtrate.

Rf in BuzPyzAczWa (4:3/4:l/4:1)=0.22 on SiO (3) In the same manner areprepared the acetates of:

H-fl-Ala-Glu-D-His-Phe-Lys-Tra H-Val-Glu-D-His-Phe-Lys-TraH-Ala-Glu-D-His-Phe-Lys-Tra Desamino-Met-Glu-D-His-Phe-Lys-Tra.

EXAMPLE XIII H-Met-Glu-His-Phe-Lys-Phe-OH and derivatives '(1) Boc MetGlu(OtBu)-D-HisPhe-Lys(Boc)-Phe- OH:

Starting from the Boc-Met-Glu(OtBu)-D-His-N pre pared in Example IX.1(9.35 g. of hydrazide converted into azide and diluted to 50 ml. of aDMF solution), Ms part is coupled to 3.2 mmol H-Phe-Lys(Boc)-Phe-OH(prepared in 0.4). The reaction mixture is adjusted to pH 7.3 with TAAand stirred at 0 C. for hours. After acidifying to pH 4, the DMFsuspension is poured into the eightfold quantity of water.

The resulting precipitate is stirred at 0 C. for another 3 hours andfiltered off.

Yield: 1.63 g. of peptide; meling point 188 dec.

(2) Boc Met Glu(OtBu)-D-His-Phe-Lys(Boc)-Phe- NHgI The azide /5 part ofthe solution mentioned in (1) is coupled to 3.2 mmolHPhe-Lys(Boc)-Phe-NH (0.3) and processed in the same way as in l, butwithout being acidified.

Melting point 204 dec. Brown colouring.

(3) Boc Met Glu(OtBu)-D-His-PheLys(Boc)-Phe- OMe:

A; part of the azide solution prepared in (1) is coupled to 3.2 mmolH-Phe-Lys(Boc)-Phe-'OMe (0.2) and processed in the same way as describedin 1, Without being acidified.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:l)=0.6l on SiO (4) Boc- Met-Glu OtBu)-D-His-Phe-D-Lys (Boc) -Phe OtBu:

part of the azide solution prepared in (1) is added to a solution of 3.2mmol H-Phe-D-Lys(Boc)-Phe-OtBu in 10 ml. of DMF (R4). The pH is adjustedto 7.3 with TAA, after which the reaction mixture is stirred at 0 C. for70 hours and then evaporated in vacuum. The residue is taken up inaqueous ethyl acetate and washed 3 times with water, after which theethyl acetate layer is dried and evaporated. The residue isrecrystallized from alcohol-ether/petroleum ether 1:1). Yield: 2.1 g.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1)=0.73 on Si03- (5)Boc-Met-Glu(OtBu)-D-His-Phe-Lys(Boc)-PPA:

/s part of the solution prepared in (1) is added to the cooled solutionof 3.3 mmol of H-Phe-Lys(Boc)-PPA (Q4) in 10 ml. of DMF. The pH isadjusted to 7.2, after which the reaction mixture is stirred for 70hours and then processed in a conventional manner. The PPA- peptide isrecrystatllized from dry ethyl acetate.

R in To:EtOH (8:2)=0.35 on SiO (6) Treatment of the peptides obtained in1-5:

Of the peptides described above 0.5 g. is dissolved in 25 ml. of TFA,stirred for 1 hour and then processed further in the usual manner. Afterthat the exchange for acetate takes place in 50 ml. of t-BuOH:H O (1:1).

Obtained in this way the acetates of:

H-Met-Glu-D-His-Phe-Lys-E9A: Rf 0.3

*Rf in Bu :Py :Ac :Wa (4 :3/4 :1/4 :1) on Si02.

EXAMPLE XIV H-Met-Glu-D-His-Phe-Lys-Phe-Gly-OH Boc-Met-Glu(OtBu)-D-His-NH (3.74 g.) is converted into the azide (see Example IX.1) and coupledto 6.4 mmol H-Phe-Lys(Boc)-Phe-Gly-OH (R3) in the manner described inExample XIII.1.

The reaction mixture obtained is poured into water and the resultingprecipitate filtered off.

R1 in BuzPyrAczWa (4:3/4:1/4:l)=0.50 on SiO Treatment as described inExample 1.2 gives the heptapeptide acetate.

Rf in BuzPyzAczWa (2:3/4:l/4:l):0.25 on SiO EXAMPLE XVI-I-A-Glu-D-I-Iis-Phe-Lys-Phe-OH In the manner described in ExampleXIII.1 the hydrazides, prepared in D1 are converted into the azide andcoupled to the peptide derivative prepared in 0.4 or P.4. The peptidesobtained are treated in the manner described in Example XIII.6.

The following peptides are obtained as acetic acid salts:

H-B-Ala-Glu-D-I-Iis-Phe-Lys-Phe-OH H-Gly-Glu-D-His-Phe-Lys-Phe-OH H-a-Me) Ala-Glu-D-His-Phe-Lys-Phe-OH H-Val-Glu-D-I-Iis-Phe-Lys-Phe-OHH-Ala-Glu-D-His-Phe-LysPhe-OH Desamino-Met-Glu-D-His-Phe-Lys-Phe-OHDesamino-Met-Glu-D-His-Phe-D-Lys-Phe-OH.

EXAMPLE XVI Sulfoxides of Metand Desamino-Met peptides 0.06 mmol of the(Met-containing) peptide is dissolved in 5 ml. of acetic acid, afterwhich ,ul. of 30% hydrogene peroxide are added. The mixture is stirredat C. for 1 hour, after which a suspension of 20 mg. of platinum (black)in 2.5 ml. of glacial acetic acid is added. Then the mixture is stirredfor 30 minutes and filtered, and the solvent distilled off in vacuum.The resulting residue is taken up in 10 ml. of tert. butanol/ water andlyophilized.

Obtained in this way:

Rf*:0.22 Desamino-Met (e O -Glu-D-His-Phe-Lys-Tra: Rf 0.2

*Rf in Bu :Py :Ac :\Va. (4 23/4 21/4 :1).

EXAMPLE XVII Sulfones of Metand Desamino-Met peptides 0.2 mmol of the(Met-containing) peptide is dissolved in a mixture of 0.5 ml. of water,0.1 ml. of 4 N perchloric acid, 0.02 ml. of 0.5 M ammoniummolybdate,after which 0.06 ml. of 30% hydrogen peroxide is added. The mixture isstirred for 2 hours at a temperature of about 1015 C. and then dilutedwith 25 ml. of tbutanol/water. After that Dowex X8 in the acetate formis added. The mixture is stirred for minutes, after which is is filteredand the resulting filtrate is lyophilized.

Obtained in this way the acetates of:

Met(+O )-Glu-D-His-Phe-Lys-Tra: Rf*=0.20 Met(O)-Glu-D-His-Phe-Lys-Phe-OH: R *=0.16 Met(+O)-Glu-D-I-Iis-Phe-D-Lys-Phe-OH: Rf 0.25 Met(+ O-Glu-D-His-Phe-Lys-Phe-Gly-OH: RV 0.22

Desamino-Met( O )-Glu-D-His-Phe-Lys-Phe-OH:

Desarnino-Met 0 -Glu-D-His-Phe-Lys-Tra:

H-Met( O )-D-Glu-His-Phe-Arg-Tra: Rf*=0.16

*Rf in Bu :PyzAczWa (413/421/421) on SiOz. **Rf in Bu :Ac :Wa (4 :1 :1)on S102.

EXAMPLE XVIII Zinccomplexes Of a solution of zinc chloride, containing50 mg. of zinc per ml., 1.5 ml. are added to a solution of 31.5 mg. ofNa HPO .2H O in 30 ml. of distilled water. The precipitate of zincphosphate formed is dissolved again by adding 4 N HCl. Then mg. of NaCland 0.5 g. of benzylalcohol are added to this mixture. Then 1.5 mg. ofthe hexapeptide H-L-Met-L-Glu-D-His-L-Phe-L-Lys- LPhe-OH (ExampleXIII.6) are dissolved in this mixture and then enough 1 N sodiumhydroxide to adjust the pH of the mixture to 8.5. After that the volumeis completed to 50 ml. with distilled water.

One ml. of suspension contains:

30 g. of hexapeptide 1.5 mg. of zinc mg. of N21 HPO 2H O 3.5 mg. of NaCl10 mg. of benzylalcohol What is claimed is: 1. A peptide of the formula:

in which one of the amino radicals Glu(Q) or His is present in theD-form; in which A is selected from the group consisting of H-L-Met,

H-L-Met(- O), H-L-Met( O desamino-Met, desamino-Met( 0), desamino-Met( Oand the moiety: H N-B-CO in which B is alkylene having 1-6 carbon atoms;Q is selected from the group consisting of OH and NHL; X is selectedfrom the group consisting of hydroxy;

(N-phenylalkyl) amino of the formula in which Alk is alkylene with 16carbon atoms, and R is selected from the group consisting of hydrogenand hydroxy; and -L-Phe-Y;

Y is selected from the group consisting of hydroxy, descarboxyl-lysyl,descarboxy arginyl, -L-Lys-Z, -L-Arg-Z, -D-Lys-Z, and -D-Arg-Z;

Z is selected from the group consisting of hydroxy,

(N-fi-indolyl-ethyl) amino, -LTrp-OH,

-L-Phe-OH, -L-Phe-Gly-OH, and (N-phenylalkyl) amino of the formula inwhich Alk is alkylene with 1-6 carbon atoms, R

is selected from the group consisting of hydrogen and hydroxy; andfunctional derivatives of said peptide selected from the groupconsisting of pharmaceutically acceptable acid addition salts,derivatives in which one or more free amino groups are substituted byacyl derived from an aliphatic carboxylic acid with 1-6 Carbon atoms,unsubstituted amides or lower alkyl (1-6 C) substituted amides of thosepeptides having a free carboxyl group, esters de- 25 rived fromaliphatic or phenylaliphatic alcohols with 1-18 carbon atoms, and metalcomplexes thereof.

2. A peptide of the formula:

A-L-Glu(Q)-D-His-X in which A is selected from the group consisting ofH-L-Met,

H-L-Met( H-L-Met( O desamino-Met, desamino-Met( O), desamino-Met(- O andthe moiety: HgN-B-CO- in which B is alkylene having 1-6 carbon atoms; Qis selected from the Group consisting of OH and NH X is selected fromthe group consisting of hydroxy, (N-

phenylalkyl) amino of the formula in which Alk is alkylene with 16carbon atoms, and R is selected from the group consisting of hydrogenand hydroxy; and -L-Phe-Y;

Y is selected from the group consisting of hydroxy, descarboxyl-lysyl,descarboxy arginyl, -L-Lys-Z, -L-Arg-Z, -D-Lys-Z, and -D-Arg-Z;

Z is selected from the group consisting of hydroxy, (N B indolyl ethyl)amino, -L-Trp-OH, -L-Trp-Gly-OH, -L-Phe-OH, -Phe-Gly-OH, and (N-phenylalkyl) amino of the formula -L-Phe-OH, -L-Phe-G1y-OH, and(N-phenylalkyl) amino of the formula 2 in which Alk is alkylene with 1-6carbon atoms, and R is selected from the group consisting of hydrogenand hydroxy. 4. A peptide of the formula:

A-Glu(Q)-His-X in which A is selected from the group consisting ofdesamino-Met, desamino-Met(- O),

desamino-Met 0 and fi-Ala, and one of the amino acid radicals Glu(Q) orHis is present in the D-form;

Q is selected from the group consisting of OH and NH X is selected fromthe group consisting of hydroxy,

(N-phenylalkyl) amino of the formula R1 -NHA1 3 in which Alk is alkylenewith. 1-6 carbon atoms, and R is selected from the group consisting ofhydrogen and hydroxy; and -L-Phe-Y;

Y is selected from the group consisting of hydroxy, descarboxyl lysyl,descarboxyl arginyl, -L-Lys-Z, -D-Lys-Z, -L-Arg-Z and -D-Arg-Z;

Z is selected from the group consisting of hydroxy,

(N-[3-indolyl-ethyl) amino, -L-Trp-OH,

-L-Trp-Gly-OH,

-L-Phe-OH, -Phe-Gly-OH, and (N phenylalkyl) amino of the formula t-NH-Alk 3 in which Alk is alkylene with 1-6 carbon atoms, and R isselected from the group consisting of hydrogen and hydroxy.

5. Metal complexes of the peptides and peptide derivatives as claimed inclaim 1.

References Cited UNITED STATES PATENTS 3,479,333 11/1969 Greven 260112.53,632,743 1/ 1972 Geller et a1. 26O---ll2.5 3,228,927 1/ 1966 Kappeleret a1 260l12.5

LEWIS GOTTS, Primary Examiner R. J. SUYAT, Assistant Examiner US. Cl.X.R. 424-177, 179

1. A PEPTIDE OF THE CORMULA: